Campylobacter jejuni is the most common bacterial cause of diarrheal disease of humans in the United States. It has recently also been implicated as one of the most common infections to precede the development of Guillain-Barre syndrome. The study of C. jejuni pathogenesis has not yet identified many major virulence factors; in particular, almost nothing is known about the contribution of C. jejuni to disease. The broad long term goal of this proposal are to characterize the interactions of a C. jejuni toxin, called cytolethal distending toxin (CDT) with eukaryotic cells, and to determine the role of CDT in human disease. To date, CDT is the only toxin produced by C. jejuni which has actually been proven to exist. CDT blocks eukaryotic cells in the G2 phase of the cell cycle, an action unlike that of any other known bacterial toxin; and, the predicted amino acid sequence of CDT proteins share no homology with the amino acid sequences of any proteins in the database. The specific aims of the proposal are designed to test the hypothesis that CDT is a virulence factor in C. jejuni diarrheal disease by virtue of its ability to block eukaryotic cells in the G2 phase of the cell cycle. Under aim 1, the ability of the CDT to cause a G2 block in cultured cell line derived from intestinal epithelial cells will be tested. In addition, Dr. Pickett will test whether CDT is active against the cell lines when they are relatively undifferentiated or still proliferating. Under aim 2, the initial interactions of CDT with HeLa cells will be characterized and experiments will be performed to determine if all or a portion of CDT enter the cells. In aim 3, the nature of the G2 block caused by CDT will be examined in greater detail. Under aim 4, the ability of CDT to contribute to C. jejuni diarrheal disease in a piglet model will be tested.